Systems and methods for multiplex analysis of PCR in real time

a real-time, multiplexing technology, applied in the field of systems and methods, can solve the problems of limited difficulty in quantifying the starting template, and unparallel amplification and precision capability, and achieve the effect of increasing the multiplexing capability of real-time pcr

Active Publication Date: 2008-12-11
LUMINEX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008]The present invention relates to systems and methods for amplification and detection of DNA. In particular, the present invention provides systems and methods that greatly increase multiplexing capabilities of real-time PCR. In one embodiment, the present invention provides a method of amplifying and detecting a plurality of nucleic acid targets in a sample comprising: (a) combining in a chamber a sample comprising the plurality of nucleic acid targets, a plurality of primer pairs for priming amplification of the plurality of nucleic acid targets, a labeling agent, and a plurality of probes complementary to the plurality of nucleic acid targets, wherein the probes are immobilized on a plurality of encoded particles such that the identity of each probe is known from the encoded particle on which it is immobilized; (b) performing an amplification cycle to form amplification products for each of the plurality of nucleic acid targets amplified with the plurality of primer p...

Problems solved by technology

PCR has been accepted by molecular biologists as the method of choice for nucleic acid detection because of its unparalleled amplification and precision capability.
DNA detection is typically performed at the end-point, or plateau phase of the PCR reaction, making it diffic...

Method used

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  • Systems and methods for multiplex analysis of PCR in real time
  • Systems and methods for multiplex analysis of PCR in real time
  • Systems and methods for multiplex analysis of PCR in real time

Examples

Experimental program
Comparison scheme
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example 1

1. Example 1

The Stability of Magnetic Microspheres Under PCR Cycling Conditions

[0184]This study demonstrates the robust stability of the Luminex super paramagnetic microspheres in PCR cycling conditions. It also demonstrates that an amplicon can be captured and detected at various stages of the PCR reaction as the concentration thereof increases. This study also shows that a two-probe system is a viable design for real-time PCR detection using magnetic microspheres. Hybridization probes (US 2001 / 6174670) also use a two-probe system.

[0185]Experimental Design: A two probe system was used to detect the generation of a Factor V gene amplicon while in the presence of magnetic microspheres that were coupled with target specific nucleotide probes. A target specific probe (FV Probe Ano) was attached to bead set 22. This probe was not fluorescently labeled. Another probe was included in the mix, which was not attached to a bead set, but was target specific, and labeled with a Cy3 fluorophore...

example 2

2. Example 2

Amplification Detection Using Tagged Primers

[0223]The following example demonstrates that a nucleic acid amplification signal can be measured in real-time using the tagged primer method. This example also demonstrates that these measurements can be performed using a imaging system comprising a quartz imaging chamber and a magnet that can be moved adjacent to the quartz imaging chamber to magnetically pull the superparamagnetic particles to a two dimensional surface of the chamber opposite a charge coupled device (CCD) detector (see e.g., FIGS. 1 and 2) in the presence of, and without modification to, the Polymerase Chain Reaction solution, and that the measurements thereof are comparable to the Luminex 200 system. The imaging system comprising the quartz chamber and magnet may be considered a “static” imaging system since the detector takes an image of the particles and any molecules bound to them while they are immobilized on the surface of the chamber by the magnetic f...

example 3

3. Example 3

Multiplex of 8

[0238]In this example, the Direct Hybridization method was used with 8 primer sets and 14 bead sets. An excess of bead sets were used to show that the amplification products do not non-specifically hybridize to bead sets containing unrelated probes.

[0239]A PCR cocktail was made including the following reagents:

TABLE 41x vol. μL45Qiagen hotstart plus 2xMaster251125MixH2O Ambion nuclease freeH2O177658 plex ordered from IDTPrimer145Coriell Sample 13033Template145Luminex MagPlexBeads3135microspheres50 mM mgCl2 Qiagen3135total502250

[0240]This cocktail, including the Luminex MagPlex microspheres with probe sequences attached thereto, was aliquoted into 40 PCR tubes at 50 μL each. Then 20 of the reactions had approximately 100 ng of genomic DNA added to them. These reactions were cycled on a BioRad iCycler thermal cycler. The primers were designed to amplify specific regions of the cystic fibrosis CFTR gene. Primer sequences for each of 8 primer sets used for this...

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Abstract

The present invention provides methods and systems for real-time measurements of PCR with multiplexing capability. Certain embodiments relate to methods and systems that use fluorescently encoded superparamagnetic microspheres for the immobilization of amplification products during the PCR process, and an imaging chamber of a measurement device that is also capable of controllable thermal cycling for assisting the PCR process.

Description

[0001]This application claims priority to U.S. provisional patent application 60 / 869,742, filed Dec. 13, 2006, the entire disclosure of which is incorporated by reference.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]This invention generally relates to systems and methods for performing measurements of DNA amplification such as PCR. In particular, this invention relates to “real time” measurements of PCR with multiplexing capability. Certain embodiments relate to a system which uses particles, such as paramagnetic microspheres, and an imaging chamber of a measurement device which is also capable of controllable thermal cycling for assisting the PCR process.[0004]2. Description of Related Art[0005]Polymerase chain reaction (PCR) is a molecular biology technique for enzymatically replicating DNA without using a living organism. PCR is commonly used in medical and biological research labs for a variety of tasks, such as the detection of hereditary diseases, the identi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6818C12Q1/6834C12Q1/686C12Q1/6804C12Q2531/113C12Q2561/113C12Q2563/143C12Q2537/143C12Q2563/107C12Q1/6837
Inventor WHITMAN, DOUGLAS F.COLLINS, CHARLES J.
Owner LUMINEX
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